Sphingolipids: An Enigmatic Species of Cell Signaling Lipids
The term sphingolipids dates back to 1884 when the German pathologist and “father of neurochemistry” Johann Ludwig Wilhelm Thudichum (1829-1901) first described a class of new lipids derived from the base sphingosine (FIG. 1A) [1]. He coined the term “sphingolipids” (from Greek “sphingos”, genitive of Sphinx), suggesting they were mysterious as the Sphinx herself. To date, more than hundred different sphingolipids are known. They are essential components of cellular membranes and have been implicated in a variety of biological functions (FIG. 1B). Among these, their roles as pro- or anti-apoptotic and pro- or anti-proliferative signaling lipids are the most important. To establish a profile of these functions for individual sphingolipids is difficult because of their rapid metabolic interconversion (FIG. 1B).
Ceramide is a membrane-resident sphingolipid and metabolic precursor for sphingosine, sphingosine-1-phosphate (S1P), ceramide-1-phosphate, sphingomyelin, and glycosphingolipids (FIGS. 1A and B). In addition to being important in stabilizing cellular membranes, sphingolipids have emerged as second messenger lipids in cell signaling pathways that regulate apoptosis, cell polarity, and differentiation. The ability of sphingolipids to form lipid microdomains or rafts determines their unique role as interface between extracellular growth factors or cytokines, and intracellular cell signaling pathways. Of particular significance is the enzymatic conversion of sphingomyelin to ceramide in the cell membrane, which is triggered by pro-apoptotic cytokines (FIG. 1A).
A major drawback in understanding the function of sphingolipids is that it is mostly not known with which proteins they interact. Binding partners and even specific binding domains have been identified for many other signaling lipids. For example, diacylglycerol, an important pro-proliferative lipid, interacts with the C1 domain of classical protein kinase C (PKCα) and protein kinase D (PKD) [15-19]. Phophoinositols, another class of signaling lipids, bind to the pleckstrin homology (PH) domain [19, 20]. Once a protein domain of this type has been identified it can be predicted from the amino acid sequence that the protein will bind to the cognate lipid. For sphingolipids, binding partners have only been specified for ceramide [2, 8, 21-30], ceramide-1-phosphate [31-33], S1P [34-37], and some gangliosides (e.g. GM1). In most cases) it is still not clear, which conserved protein domain will interact with a particular sphingolipid.
The “classical” interaction partners of ceramide are ceramide-activated protein phosphatase(s) [22, 23, 38] and kinase(s) [8, 24, 25, 27, 28, 30, 39-43]. Recent studies have shown that ceramide activation of protein phosphatase 1 (PP1) alters splicing of B-cell lymphoma X (bcl-x) and caspase 9 from anti- to pro-apoptotic proteins [44]. Ceramide-activated protein phosphatase 2A (PP2A) is involved in de-phosphorylation of a variety of key factors in cell signaling pathways regulating proliferation, apoptosis, and differentiation [22, 38, 45].
Within the group of protein kinases, kinase suppressor of Ras (KSR) [46, 47], PKCδ [42, 43], and aPKC [2, 3, 8, 24-28, 45] have been found to be activated by ceramide. In the last ten years, activation of aPKC by ceramide was independently confirmed by several groups [2, 8, 24-28]. A relative of the C1 domain, the C1B domain was identified in the amino acid sequence of PKCδ and aPKC. Because of its structural similarity to diacylglycerol, ceramide has been suggested to bind to this domain [15, 40-42]. It was not until recently, however, that evidence for direct binding of aPKC to ceramide was found [2, 8, 24, 26, 27]. These studies have focused on aPKC based on its affinity to ceramide and novel ceramide analogs [2,8]. Ceramide and most likely many ceramide analogs form organized lipid microdomains or rafts in the cell membrane [26, 48-54]. These rafts may allow for repeated and multiple binding (avidity) of ceramide-associated proteins, thereby enhancing the formation of protein complexes with cell signaling functions. Therefore, it is tempting to speculate that ceramide-induced rafts and associated protein complexes form an initial platform for growth factor or cytokine-dependent cell signaling pathways.
Lipid Rafts, and Sphingolipid-Induced Protein Scaffolds (SLIPS): A Platform for Cell Signaling Pathways
Three major cell signaling pathways are regulated by cytokines and growth factors that activate sphingomyelinases, a group of acidic or neutral pH-dependent enzymes that elevate the ceramide concentration in the cell membrane by catalyzing the hydrolysis of sphingomyelin (FIG. 1A). Two of these cell signaling pathways, CD95/FasL [52, 53, 55-58] and TNFα [30, 46, 48, 59-62], are known to induce apoptosis in a variety of cell types by the activation of acid sphingomyelinase (ASMase). In contrast to the cytokine-activated receptors, the p75NTR cell signaling pathway is not a priori pro-apoptotic. The neurotrophin receptor p75NTR is expressed by many neural cell types and induces axonal outgrowth in the peripheral nervous system when stimulated with nerve growth factor (NGF) [63-67].
However, evidence has amounted that p75NTR-induced apoptosis is a major factor in neurodegenerative diseases such as Alzheimer's disease [68-76]. This apparently paradoxical, dual function of p75NTR, pro-apoptotic or pro-outgrowth, has been explained by a model suggesting that the effect of p75NTR activation depends on heterodimerization with other neurotrophin receptors [77-87]. If p75NTR forms heterodimers with tropomyosin-related kinase A (trkA), a tyrosine kinase receptor, activation of the chimeric receptor induces axonal outgrowth. However, if p75NTR forms homodimers binding of NGF to p75NTR activates neutral sphingomyelinase (NSMase), which then generates ceramide and may induce apoptosis.
For a long time, ceramide was stigmatized as being an exclusive inducer of apoptosis. This bias resulted mainly from experiments that used a short chain analog of ceramide, N-acetyl sphingosine (C2 ceramide) to test the induction of apoptosis by ceramide. C2 ceramide is ideally suited as medium supplement because its water solubility is several-fold higher than that of physiological ceramide species (e.g., N-palmitoyl sphingosine or C16 ceramide). However, recent advances in administering ceramide with long fatty acid chains and the development of novel ceramide analogs has clearly shown that ceramide has additional, non-apoptotic functions [2, 3, 7, 8, 88-92]. In particular, it has demonstrated that the pro- or non-apoptotic function of ceramide depends on effectors that modulate the activation of ceramide-associated proteins [2-5].
Ceramide has been shown to form microdomains or rafts within cellular or synthetic membranes [26, 41, 48, 51, 54, 56, 93]. Lipid rafts are originally characterized by being insoluble in detergent. Using this unique feature to isolate rafts many membrane-resident proteins have been characterized as being raft or non-raft proteins. Unfortunately, it has turned out to be difficult to directly visualize these rafts, which is important to show their biological significance. One of the main reasons for this shortcoming was the unavailability of antibodies against membrane lipids. With respect to ceramide, this has been tremendously improved within the last couple of years [94, 95]. For example, a novel antibody against ceramide has been developed that was used to determine the polarized distribution of ceramide in membrane protrusions of neural cells and apical cell membranes of primitive ectoderm cells [3, 95].
How would the polarized distribution of ceramide in lipid rafts support its function as second messenger lipid for cell signaling pathways? Ceramide is mainly distributed to three compartments of the cell. De novo biosynthesis of ceramide from serine and plamitoyl-CoA takes place in the endoplasmic reticulum (ER) [96-99]. The hydrophobic alkyl chain of sphingosine and the fatty acid residue are buried within the membrane, while the polar head group of the sphingosine (serine) portion faces the cytosol. From the ER, ceramide is transported to the Golgi via ceramide transport protein (CERT) [97, 100]. In the Golgi, ceramide is derivatized by attaching phosphorylcholine or glyosyl groups, which generates sphingomyelin or glycosphingolipids, respectively. At this point, the polar head group has flipped from the cytosolic to the lumenal part of the Golgi. Sphingomyelin and glycosphingolipids are transported to the cell membrane, the polar head group facing the outside of the cell. It becomes clear that any association of ceramide with cytosolic proteins will first require flipping the polar head group back to the inside of the cell.
It should be noted that other compartments, in particular mitochondria, the nucleus, and lysosomes contain ceramide pools as well. Ceramide has been suggested to open a mitochondrial transition pore, which releases pro-apoptotic proteins such as cytochrome c and apoptosis inducing factor (AIF) [101, 102]. In the nucleus, ceramide could affect the alternative splicing of RNA encoding pro- or anti-apoptotic proteins, or cause an imbalance of calcium levels [103, 104]. The lysosomes are known to generate ceramide via activation of acid sphingomyelinase, an enzyme affected in Niemann-Pick disease [105]. It has shown that elevation of ceramide in mitochondrial-associated membranes (MAM) of the ER induces a pro-apoptotic aPKC/PAR-4 complex that prevents activation of NF-κB [2]. However, it is also found that the non-apoptotic functions of ceramide are intimately linked to ceramide localized at the cell membrane.
FIG. 2A depicts a working model that shows how the localized and receptor-mediated activation of SMases generates a ceramide raft. It should be noted that ASMase is localized at the outer leaflet, while NSMase is at the inner leaflet of the cell membrane [56, 58, 106]. Accordingly, activation of ASMase generates ceramide first at the outer leaflet, which is followed by flipping of the polar ceramide head group to the inner leaflet of the membrane. In contrast to ASMase, receptor-activated generation of ceramide by NSMase will first require flipping of the polar SM head group to the inner leaflet. Once ceramide is enriched at the inner leaflet, ceramide-binding proteins such as aPKC will initiate a sphingolipid-induced protein scaffold (SLIPS), a protein complex proposed by our group for the first time [1]. A SLIPS promotes microtubule formation and as a result, protrusion of the membrane. Depending on the effect of the receptor on SMases (activating or inhibiting), binding of a growth factor or cytokine may enlarge ceramide or SM microdomains, respectively. Intriguingly, studies with synthetic model membranes have shown that ceramide and SM form microdomains that are segregated from each other [50, 51, 58, 93]. Hence, receptor activation will polarize the distribution of these two sphingolipids when the ceramide microdomain expands. Release of ceramide by SMases is an enzymatic process: receptor activation by binding of just one growth factor or cytokine molecule may generate many more ceramide molecules that organize themselves in microdomains or rafts.
It has been shown that ceramide can rapidly flip from the outside to the inside of the cell membrane [107]. Flipping of SM has been suggested to go in hand with externalization of phosphatidylserine and may involve a phospholipid binding protein termed “scramblase” or “flippase” [108-111]. Hence, accumulation of ceramide or SM in rafts at the outer membrane leaflet will quickly generate an equivalent microdomain facing the cytosol. There is indirect evidence for this “inner leaflet” microdomain coming from a recent study showing that the isolated ceramide raft fraction contains aPKC, clearly a ceramide-associated, cytosolic protein [26]. There is also evidence that “phosphatase and tensin homolog deleted on chromosome ten” (PTEN), another cytosolic protein, is associated with ceramide rafts [112]. This, however, may not involve direct binding of PTEN to ceramide but association with a protein complex organized at the ceramide raft.
Immunocytochemistry for sphingomyelin and ceramide was used to determine the distribution of these two sphingolipids in the cell membrane of neural progenitor or precursor cells (NPCs) (FIG. 2B). Although sphingomyelin and ceramide domains are in close vicinity to each other, they show only little overlap in their membrane distribution. This result is consistent with a model in that ceramide, once generated from sphingomyelin, organizes itself in separate lipid domains. Notably, ceramide is mainly distributed to a perinuclear compartment and the tip of membrane protrusions. These protrusions may represent “sphingopodia”, a term refers to the polarized distribution of ceramide in microspikes, filipodia and lamellipodia [95]. Fluorescence resonance energy transfer (FRET) was used to confirm the direct association of ceramide with aPKC in the ceramide-rich perinuclear compartment [2]. FRET is a technique that utilizes the direct, radiation-free energy transfer from one fluorophore to another one when they are close together (<10 nm). FIG. 2C shows initial studies obtaining a Cy3-to-Cy5 FRET signal from α-tubulin (bound to Cy3-conjugated antibody) to ceramide (bound to Cy5-conjugated antibody) in membrane protrusions of NPCs. In summary, these studies support the model shown in FIG. 2A in that one of the non-apoptotic functions of ceramide may be the regulation of cell polarity and assembly of microtubules.
Ceramide and S1P: Key Regulators of Stem Cell Polarity and Apoptosis
A potential non-apoptotic function of ceramide is discussed above. The following discussion focuses on the mechanism underlying this function and how it is regulated. Studies have shown that the non-apoptotic function(s) of ceramide depend on a low expression level of PAR-4, a protein that inhibits ceramide-associated aPKC. It is found that there are three stages during embryonic stem (ES) cell differentiation at which expression of PAR-4 is absent or low: undifferentiated ES cells, suspension EBs, and NPCs [2-5, 113]. Recently, it is reported that in suspension EBs, ceramide is essential for the polarity of primitive ectoderm cells [3]. Ceramide depletion prevents membrane association of aPKC, disrupts the interaction between aPKC and Cdc42, and results in decreased phosphorylation of GSK-3β. The ceramide analog S18 restores primitive ectoderm formation, indicating that it is ceramide and not one of its derivatives that regulates cell polarity, suggesting a regulatory effect of ceramide on the non-canonical Wnt or cell polarity pathway.
A working model shown in FIG. 3A explains the function of ceramide for cell polarity in NPCs and other cell types. Based on the observation that ceramide microdomains co-distribute or even associate with microtubules (FIGS. 2A and C) [1-1, 95], the effect of ceramide-associated aPKC on GSK-3β was evaluated. GSK-3β phosphorylates many proteins that regulate cell adhesion or formation of microtubules. Key factors are β-catenin, adenomatous poliposis coli (APC), and τ-protein [114-122]. Phosphorylation of β-catenin by GSK-3β renders it susceptible to proteolytic degradation [116], while hyperphosphorylation of τ causes its aggregation in tauopathy, a neurodegenerative disorder that is also involved in the etiology of Alzheimer's disease [114, 119, 120]. Phosphorylation of APC by GSK-3β disrupts its function in stabilizing the plus end of microtubules [116]. According to this model, ceramide-induced activation of aPKC results in aPKC-dependent phosphorylation and inactivation of GSK-3β. Hence, ceramide and S18 should stabilize microtubules, while ceramide depletion de-stabilizes them (FIG. 3A).
FIG. 3C shows that among the factors that regulate adherens junctions and microtubules, ceramide-activated aPKC and PP2a may complement each other. It is known that PP2a de-phosphosphorylates β-catenin, APC, and τ [115, 120]. Hence, loss of phosphorylation by ceramide-mediated inactivation of GSK-3β (via ceramide-activated aPKC) and enhanced de-phosphorylation by ceramide-activated PP2a should act synergistically on promoting the stability of microtubules. Interestingly, GSK-3β can also phosphorylate and inactivate PP2a [123]. Therefore, ceramide can activate PP2a in two ways: by direct binding to PP2a and by inactivating GSK-3β (via aPKC-mediated phosphorylation). In contrast to this, ceramide-activated PP2a may also de-phosphorylate GSK-3β, thereby antagonizing phosphorylation of GSK-3β by ceramide-activated aPKC [124].
PAR-4 is a leucine zipper protein with several functions. It was discovered by differential hybridization to identify pro-apoptotic genes expressed in androgen-dependent prostate cells [125]. Using two hybrid assays it was found to be an inhibitor of aPKC and transcriptional co-repressor of Wilms' tumor suppressor 1 (WT1) [126, 127]. Recently. PAR-4 has gained attention due to its multifaceted function in neural cells. It has been suggested to contribute to neurodegeneration in Alzheimer's and Parkinson's disease, and to the etiology of amyotrophic lateral sclerosis and stroke [86, 128-132]. In addition to its pro-apoptotic functions, PAR-4 has been shown to regulate the activity of choline acetyl transferase, to inhibit choline uptake, and to regulate synaptic plasticity [133-135]. PAR-4 has been found to be temporarily associated with the actin cytoskeleton [136]. It has also been reported that a short form of PAR-4 acts as dominant negative regulator of apoptosis by forming actin-associated heterodimers with the pro-apoptotic long form of PAR-4 [6].
When the pro-apoptotic form of PAR-4 is expressed, the non-apoptotic effect of ceramide changes fundamentally. Using lipid vesicles made of ceramide and phospholipids (termed lipid vesicle-mediated affinity chromatography or LIMAC) it was found that association of aPKC with ceramide enhances the affinity of aPKC to its inhibitor PAR-4 [2]. In the presence of PAR-4, ceramide does not activate aPKC, but on the contrary, enhances its inhibition by PAR-4 (FIG. 3B). Because of this, an initial non-apoptotic or even pro-survival function of ceramide can rapidly turn into the induction of apoptosis.
In addition to its immediate cell signaling function, ceramide serves as metabolic precursor for another important cell signaling lipid, S1P. Ceramide is hydrolyzed by ceramidase to sphingosine, which is then phosphorylated by sphingosine kinase 1 or 2 (SK 1 or 2) to S1P (FIG. 1A) [137-144]. S1P is a soluble ligand that binds and activates five isoforms of the S1P receptor [21, 34-36, 39, 145-147]. Knockout mice for SK1&2 or S1P receptors have clearly demonstrated the essential function of S1P for vascular and neural development [138]. Recent studies indicate that one of the functions of S1P is to counterbalance ceramide-induced apoptosis [88, 141, 148]. S1P is known to increase phosphorylation of p42/44MAPK and Akt (protein kinase B), two important protein kinases that inactivate the pro-apoptotic proteins Bad and Bax (FIG. 3D) [1, 113, 145, 149-155]. Unlike S1P, ceramide has been shown to reduce the activity of p42/44-MAPK and Akt [26, 113, 154, 156]. Hence, ceramide and S1P may counter-regulate the phosphorylation of Bax and Bad, thereby controlling apoptosis and cell survival (FIG. 3D).
Pluripotency Factors and Stem Cell Derived-Tumors: Active Elimination of Risky Stem Cells with Ceramide Analogs
Embryonic stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to transplantation for treatment of diseases that result from cell damage or dysfunction. Other pluripotent cells and cell lines including early primitive ectoderm-like (EPL) cells as described in International Patent Application WO 99/53021, in vivo or in vitro derived ICM/epiblast, in vivo or in vitro derived primitive ectoderm, primordial germ cells (EG cells), teratocarcinoma cells (EC cells), and pluripotent cells derived by dedifferentiation, reprogramming or by nuclear transfer will share some or all of these properties and applications.
The ability to tightly control differentiation or form homogeneous populations of partially differentiated or terminally differentiated cells by differentiation in vitro of pluripotent cells has proved problematic. Uncontrolled differentiation produces mixtures of pluripotent stem cells and partially differentiated stem/progenitor cells corresponding to various cell lineages. When these ES-derived cell mixtures are grafted into a recipient tissue the contaminating pluripotent stem cells proliferate and differentiate to form tumors, while the partially differentiated stem and progenitor cells can further differentiate to form a mixture of inappropriate and undesired cell types.
It is well known from studies in animal models that tumors originating from contaminating pluripotent cells can cause catastrophic tissue damage and death. In addition, pluripotent cells contaminating a cell transplant can generate various inappropriate stem cell, progenitor cell and differentiated cell types in the donor without forming a tumor. These contaminating cell types can lead to the formation of inappropriate tissues within a cell transplant. These outcomes cannot be tolerated for clinical applications in humans. Therefore, uncontrolled ES cell differentiation makes the clinical use of ES-derived cells in human cell therapies impossible.
Therefore, one of the major perils using stem cells for therapy is their ability to maintain or (re-) adopt a pluripotent state that endows them with the capacity of forming teratomas (stem cell-derived tumors). Teratoma formation has been reported in roughly half of the studies using embryonic stem (ES) cell-derived stem cell grafts [5, 9, 157-167]. The risk is inversely proportional to the differentiation stage. The more differentiated the stem cells, the lower the probability of teratoma formation. However, even genetically re-programmed adult cells, regardless of initially being stem cells or differentiated cells, form teratomas if endowed with pluripotency factors [168]. Therefore, techniques are needed to prevent teratoma formation from stem cell transplants.
Teratoma formation from ES cells can be avoided by differentiating these cells to a particular progenitor stage that allows for repeated self-renewing of the progenitor cells. Continuous passaging of neural progenitors will eventually “dilute out” pluripotent cells and minimize the risk of teratomas. However, in stem cell therapy, size matters. In experiments with mice, the number of transplanted cells is usually in the range of 105-106 cells/injection. This number is pre-determined by the injection technique: the small volume of the cell suspension does simply not accommodate a larger number of cells if a single dose is injected. A human brain, however, is 1000-times larger than a mouse brain. It is questionable that this low number of cells will be able to repair tissue, in particular, if it is not desired that the cells retain the capacity of repeated cell division after transplantation. Studies have shown that within a population of embryoid body-derived cells at the stage of generating NPCs, up to 30% of the cells may retain pluripotency and therefore, pose a serious risk of teratoma formation [5].
Stem cell therapy without techniques actively eliminating teratoma forming cells may be successful if combined with genetic engineering of the transplanted cells. Fluorescent or surface proteins expressed under the control of a progenitor-specific promoter (e.g., nestin, Sox-1, Olig-2) have been used to “purify” NPCs or oligodendrocyte precursors by fluorescent or magnetic activated cell sorting (FACS or MACS) and to rid them of residual pluripotent cells [169-172]. Conversely, fluorescent protein expression under the control of the Oct-4 promoter can be used to remove pluripotent stem cells or to confirm loss of pluripotency in the graft. However, these methods will need stable transfection with a transgene that will be present in the graft, regardless of the gene product being expressed or not. While certainly feasible for animal studies, it will add another layer of intricacy for approval in human stem cell therapy.
Alternatively, residual pluripotent stem cells can be eliminated by harnessing an intrinsic sensitivity toward apoptosis inducers. It has been shown that these cells co-express the pluripotency marker Oct-4 and the apoptosis sensitizer PAR-4 [5]. As discussed above, PAR-4 is an inhibitor protein that binds to aPKC when associated with ceramide. Inhibition of aPKC induces apoptosis. Hence, simply incubating differentiating ES cells at the stage of forming NPCs with a ceramide analog S18 eliminates Oct-4(+) cells because they are sensitized to ceramide due to the co-expression of PAR-4. These studies have shown that this technique can be used to prevent teratoma formation when transplanting neural stem cells derived from ES cells [5].
Active elimination of teratoma forming stem cells from a graft using ceramide analogs was possible because the protein expression profile, in particular pluripotency and sensitivity to apoptosis inducers, in these cells was determined. However, it has been also found that a small portion (<5%) of useful NPCs express PAR-4, thus, are still sensitive toward ceramide. These cells, termed NPC2 cells, express the sphingosine-1-phosphate receptor 1 (S1P1) that induces anti-apoptotic cell signaling pathways when activated by binding to S1P [10-14].
There is still a need, therefore, to identify methods and compositions for the production of a population of cells enriched in neural stem cells that do not form teratomas, and the products of their further differentiation, and in particular, human neural and/or glial cells and their products.